1. Field of the Invention
This invention relates to methods for diagnosing cystic fibrosis in patients afflicted with that disease and more particularly to methods for diagnosing cystic fibrosis by detecting cystic fibrosis ciliostatic factor in body fluids of patients and carriers
2. Description of the Prior Art
Cystic fibrosis (CF) is a debilitating genetic disease characterized by abnormalities of secretory organs in the body The disease is the most common lethal genetic disease of Caucasian children, and those individuals who are diagnosed as having CF usually die before the age of twenty-five. The basic genetic defect and its mode of operation are unknown, but the defective gene is relatively common in the Causasian population, about 5% of the populace being heterozygous carriers. Since the inheritance follows the typical autosomal recessive pattern, CF homozygotes express the gene and exhibit symptoms of the disease, while CF heterozygotes are carriers of the gene but do not have symptoms of the disease.
Because the disease is life-threatening, therapy is essential. Accordingly, an accurate and reliable method of diagnosis is very important. However, the clinical picture of the disease is highly variable and clinical findings alone cannot be relied on for diagnosis. A summary of various biochemical tests for CF is found in Heeley, A. F., et al, Clin. Chem. 29, 2011-2018 (1983). The most frequently used method for CF diagnosis at present is the "sweat test", in which CF homozygotes show increased concentration of NaCl in their sweat caused by mal-reabsorption of salt. At present, there is no test for CF heterozygotes.
Other biochemical tests for CF have been based on the finding that the body fluids (blood, urine, saliva, etc.) of CF positive individuals have been found to contain abnormal substances. One of such substances which has been found to be a useful marker for cystic fibrosis is ciliostatic factor.
This substance has been given the name cystic fibrosis ciliostatic factor because it possesses the characteristic biological property of inhibiting the motion of actively moving cilia. This property is the basis of several different ciliary bioassays which have been developed to detect CF by observing the ciliostatic effect of body fluids containing CF ciliostatic factor on oyster gill cilia, mussels cilia, rabbit tracheal cilia and a number of other cilia systems. Although these bioassays are not very precise and certainly not quantitative, they can identify individuals having CF. However, because the technique is slow and difficult and requires highly trained personnel, the assays using ciliated cell systems have not been useful in a clinical setting.
Because of the difficulty of ciliated cell assays, other assays for CF ciliostatic factor have been sought. Since it was known that certain compounds which have ciliostatic activity, e.g., polyhydroxyamines, are also inhibitors of mammalian debranching enzyme, an enzyme important for breakdown of glycogen in humans, the effect of CF ciliostatic factor on this enzyme was investigated. It was found that CF ciliostatic factor is, indeed, an inhibitor of mammalian debranching enzyme, and an assay has been developed using the enzyme inhibition properties of this factor using glycogen phosphorylase limit dextrin as a substrate (Gillard, B. K., et al, J. Pediatric Res. 10, 907-910 (1976)).
An additional advantage of the inhibition assay procedure is that it appears to be able to distinguish not only homozygote CF patients from normal individuals, but also identify and distinguish heterozygote CF carriers from homozygote CF patients. However, the assay as described in the literature suffers from several deficiencies; (a) laborious sample processing must be used to rid the specimen of alpha-amylase, (b) none of the assay components are readily available from commercial sources, (c) no large scale clinical evaluation of the procedure has been carried out. The removal of alpha-amylase from the sample is necessary because the substrate used in the literature assay is also acted on by alpha-amylase. Furthermore, the purification is especially laborious because CF ciliostatic factor is bound to alpha-amylase in the body fluid (although it does not inhibit the alpha-amylase). In view of these problems, the method is not acceptable as a clinical diagnostic tool for CF. Indeed, hitherto there has been no clinically useful and practical technique for the detection of CF ciliostatic factor.
Hence a need has continued to exist for a biochemical method for analyzing for CF ciliostatic factor which is useful in clinical diagnosis of CF.